Preclinicai studies utilizing different AAV serotypes have demonstrated efficient gene transfer in animal models and stable correction of hemophilia in murine and canine models has been achieved. Early clinical trial data suggest that achievement of therapeutic levels of clotting factors is feasible in human subjects. However, the latter clinical trials have also underscored two major challenges, namely, a large patient cohort displaying pre-existing humoral immunity against AAV capsids and potential hepatotoxiclty associated with administration of high vector dose. The current proposal is focused on designing next generation AAV vectors that display potentia! to evade preexisting anti-capsid neutralizing antibodies (NAbs) and transduce human hepatocytes with high efficiency. To achieve such, we will utilize three complementary AAV capsid engineering techniques and two translational components, namely, a mouse model with humanized fiver and human serum containing anti-AAV capsid NAbs. In synergy with Project 1 of this PPG, we will first establish the hepatotropism of AAV clades with rationally altered antigenic epitopes in humanized mice. In the second and third aims, we will utilize a novel chemical engineering strategy and a combinatorial directed evolution approach, respectively to mask the antigenicity of hepatotropic AAV vectors. The proposed studies should (a) provide structural insight into the antigenicity and hepatotropism of AAV vectors, (b) humanized AAV vectors for preclinical evaluation in large animal models of hemophilia (Projects 3 & 4) and (c) optimized vector candidates for clinical trials. RELEVANCE (See instructions): Eariy clinical trial data suggest that gene therapy of bleeding disorders might be feasible in human subjects. The current proposal is focused on tackling clinically relevant challenges, namely preexisting neutralizing antibodies and the need for humanized AAV vectors validated in translational animal models. This project will enable development of new AAV vectors simultaneously capable of enhanced gene transfer to human liver and escaping nrefixistina humoral immunitv in human suhierta '.